Back to EveryPatent.com
United States Patent |
5,173,427
|
Mallonee
|
*
December 22, 1992
|
Vectors and hosts with increased expression of HBCAG
Abstract
The invention relates to vectors and hosts with increased expression of
HBcAg.
Inventors:
|
Mallonee; Richard L. (Catonsville, MD)
|
Assignee:
|
Becton Dickinson and Company (Franklink Lakes, NJ)
|
[*] Notice: |
The portion of the term of this patent subsequent to December 29, 2009
has been disclaimed. |
Appl. No.:
|
739642 |
Filed:
|
August 1, 1991 |
Current U.S. Class: |
435/252.33; 435/252.3; 435/320.1 |
Intern'l Class: |
C12N 001/21; C12N 015/70 |
Field of Search: |
435/69.3,172.1,252.3,252.33,320.1,240.2
935/22,29,33,38,39,66,73
|
References Cited
U.S. Patent Documents
4710463 | Dec., 1987 | Murray | 435/69.
|
Foreign Patent Documents |
0182442 | May., 1986 | EP.
| |
0271302 | Jun., 1988 | EP.
| |
0272483 | Jun., 1988 | EP.
| |
0304238 | Feb., 1989 | EP.
| |
Other References
Yie et al., "High Level of Expression of HBcAg in E. coli by modification
of the 5'end of the HBc Gene" Chinese Journal of Virology 4(4):312-318
(1988).
Kim et al., "Expression and Secretion of Hepatitis B Viral Mutant Core
Antigen" Korean Journal of Microbiology 27(3):169-175. (1989).
Grosjean et al., "Preferential codon usage in prokaryotic genes: the
optimal codon-anticodon interaction energy and the selective codon usage
in efficiently expressed genes" Gene 18: 199-209 (1982).
R. A. Bhat et al., Hepatology 11:271 (1989).
R. E. Lanford et al., Viral Immunology 1:97 (1987).
|
Primary Examiner: Schwartz; Richard A.
Assistant Examiner: Johnson; Michelle
Attorney, Agent or Firm: Stierwalt; Brian K.
Claims
What is claimed is:
1. A vector comprising a DNA molecule having a sequence consisting
essentially of Sequence ID No: 3, Sequence ID No: 5, Sequence ID No: 7,
Sequence ID No: 9, or Sequence ID No: 11.
2. The vector of claim 1 in which said sequences further comprises about an
8 to 20 base pair spacer before the initiation codon.
3. The vector of claim 2 in which said spacer consists essentially of
Sequence ID No: 13.
4. The vector of claim 2 in which said spacer consists essentially of
Sequence ID No: 14.
5. The vector of claim 1 in which the second codon of said sequence is a
host preferred codon.
6. The vector of claim 2 in which the second codon of said sequence is a
host preferred codon.
7. The vector of claim 3 in which the second codon of said sequence is a
host preferred codon.
8. The vector of claim 4 in which the second codon of said sequence is a
host preferred codon.
9. The vector of claim 5 in which said host preferred codon is an E. coli
preferred codon.
10. The vector of claim 6 in which said host preferred codon is an E. coli
preferred codon.
11. The vector of claim 7 in which said host preferred codon is an E. coli
preferred codon.
12. The vector of claim 8 in which said host preferred codon is an E. coli
preferred codon.
13. A host transformed with a vector comprising a DNA molecule having the
sequence consisting essentially of Sequence ID No: 3, Sequence ID No: 5,
Sequence ID No: 7, Sequence ID No: 9, or Sequence ID No: 11.
14. The host of claim 13 in which the vector further comprises about an 8
to 20 base pair spacer before the initiation codon of said sequences.
15. The host of claim 14 in which said spacer consists essentially of
Sequence ID No: 13.
16. The host of claim 14 in which said spacer consists essentially of
Sequence ID No. 14.
17. The host of claim 13 in which the second codon of said sequences are
host preferred codons.
18. The host of claim 14 in which the second codon of said sequences are
host preferred codons.
19. The host of claim 15 in which the second codon of said sequences are
host preferred codons.
20. The host of claim 16 in which the second codon of said sequences are
host preferred codons.
21. The host of claim 13 which is E. coli.
22. The host of claim 14 which is E. coli.
23. The host of claim 15 which is E. coli.
24. The host of claim 16 which is E. coli.
Description
FIELD OF THE INVENTION
The invention is in the field of molecular biology. In particular, the
invention relates to new sequences and improved expression of hepatitis B
core protein.
BACKGROUND OF THE INVENTION
Hepatitis B virus is the most thoroughly characterized pathogen of the
hepatitis diseases. The hepatitis B virus is associated with a wide
spectrum of liver disease, from a subclinical carrier state to accute
hepatitis, chronic hepatitis, postnecrotic (posthepatitic) cirrhosis, and
hepatocellular carcinoma. It also has a poorly understood association with
several primary non-hepatic disorders including polyarteritis nodesa and
other collagen vascular diseases, membraneous glomerulonephritis,
essential mixed cryoglobulinemina and papular acrodermatitis of childhood.
Hepatitis symptoms and signs vary from minor flu-like illnesses to
fulminant, fatal liver failure.
Groups at risk for contracting hepatitis B virus include certain hospital
and dentist staff (e.g., oncology, hemodialysis-transplantation,
gastroenterology, intensive care units, diagnostic laboratories, and
surgical units), staff in institutions for the mentally handicapped,
patients receiving blood and blood products, drug addicts, male
homosexuals, and the families of chronic carriers.
The infective "DANE" particle consists of an inner core plus an outer
surface coat. The inner core contains DNA and DNA polymerase. The DNA
replicates within the nuclei of infected hepatocytes. The core antigen
(HBcAg) is associated with the viral inner core. It can be found in
infected liver cells but is not detectable in serum except by special
techniques which disrupt the DANE particle. The hepatitis B virus is
present in the cytoplasm of parenchymal liver cells of individuals with
hepatitis B and constitutes the infective virus. The core particle
displays HBcAg. The core of this particle is found in the nucleus of
parenchymal cells, but as it passes through the cytoplasm, it acquires a
surface coat.
Antibody to the core antigen appears promptly in the blood of infected
individuals and persists indefinitely. High titers of IgM anti-HBc is
found in patients with accute disease and may be the only marker of accute
hepatitis B in some situations.
Serological detection of anti-HBc is accepted diagnostic evidence of
hepatitis B viral infection. Therefore, it is desirable to have
substantial quantities of HBcAg for use as an immunogen in development of
monoclonal and polyclonal antibodies to the HBcAg, for preparing vaccines,
and for use in detection of the viral infection in patients.
SUMMARY OF THE INVENTION
The present invention provides new HBcAg sequences with improved
expression. The invention also provides nucleotide sequences which enhance
expression of HBcAg. The invention also provides new HBcAg protein
sequences. In addition, the invention provides methods for increasing
expression of HBcAg.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 Enzyme linked immunosorbent Assay (ELISA) data showing increased
expression of HBcAg with sequences of the invention.
DETAILED DESCRIPTION
The present invention provides new HBcAg sequences and methods for
increasing expression of HBcAg. When Sequence ID No: 1 is altered to
include additional nucleotides between the ribosome binding site and start
codon, (also referred to as the initiation codon), the expression level is
about ten (10) times higher than the same sequence unmodified. When the
modification involves changing the second codon (first codon after the
start codon) from GAC to GCT, plus nucleotides between the ribosome
binding site and first ATG codon, increases of about twenty-five (25)
times higher expression are obtained over the native sequence.
The present invention also provides DNA molecules described in Sequence ID
No: 3, Sequence ID No: 5, Sequence ID No: 7, Sequence ID No: 9, and
Sequence ID No: 11.
The invention also provides hosts, and methods for increasing expression of
HBcAg which comprises transforming a host with the disclosed sequences.
The invention also provides amino acid sequences described in Sequence ID
No: 2, Sequence ID No: 4, Sequence ID No: 6, Sequence ID No: 8, Sequence
ID No: 10, and Sequence ID No: 12 and kits comprising the sequences.
Sequence ID No: 1 is referred to as the "native" sequence. Preferably
Sequence ID No: 1 has an eight base pair spacer between the ribosome
binding site and start codon. Most preferably the eight base pair spacer
is Sequence ID No: 14 (AACAGACC). Sequence ID No: 3 is the same as
Sequence ID No: 1 (silent mutations from a G to A at bp 39 and a C to T at
bp 249 are present). Preferably Sequence ID No: 3 has a 10 base pair
spacer between the ribosome binding site and the start codon. Most
preferably the ten base pair spacer is Sequence ID No: 13 (AACAGAATTC).
Sequence ID No: 5 is the same as Sequence ID No: 1 but has the alterations
of the second codon change, bp 467 changes from G to A and deletion of bp
468, causing a frameshift. Preferably Sequence ID No: 5 has a ten base
pair spacer between the ribosome binding site and start codon. Most
preferably the ten base pair spacer is Sequence ID No: 13. Sequence ID No:
7 is the same as Sequence ID No: 1 but has the second codon change.
Preferably Sequence ID No: 7 has an eight base pair spacer between the
ribosome binding site and start codon. Most preferably the eight base pair
spacer is Sequence ID No: 14. Sequence ID No: 9 is the same as Sequence ID
No: 7 with a transition at bp 359 from a T to a C (resulting in a Val to
Ala change in the protein). Preferably Sequence ID No: 9 has a ten base
pair spacer between the ribosome binding site and start codon. Most
preferably the ten base pair spacer is Sequence ID No: 13. Sequence ID No:
11 is the same as Sequence ID No: 7 with alterations of the second codon
change, and deletion of bp 455, causing a frameshift. Preferably an eight
base pair spacer is between the ribosome binding site and start codon.
Most preferably the eight base pair spacer is Sequence ID No: 14.
Sequence ID No: 2, 4, 6, 8, 10, and 12 are predicted HBcAg protein
sequences of the invention. Sequence ID No: 1 encodes the protein of
Sequence ID No: 2. Sequence ID No: 3 also encodes the protein sequence in
Sequence ID No: 2 (or 4). Sequence ID No: 5 encodes the protein in
Sequence ID No: 6. Sequence ID No: 7 encodes the protein in Sequence ID
No: 8. Sequence ID No: 9 encodes the protein in Sequence ID No: 10
Sequence ID No: 11 encodes the protein in Sequence ID No: 12. Since many
amino acids are selected by more than one codon (degeneracy), DNA
sequences can vary without corresponding changes in the amino acid
sequences.
Tables 1 through 11 depict nucleotide and amino acid sequences of the
invention (nucleotide sequences left to right are 5' to 3' and amino acid
sequences left to right are amino terminus to carboxy terminus).
TABLE 1
__________________________________________________________________________
ATGGACATTG
ACCCTTATAA
AGAATTTGGA
GCTACTGTGG
AGTTACTCTC
GTTTTTGCCT
60
TCTGACTTCT
TTCCTTCCGT
CAGAGATCTC
CTAGACACCG
CCTCAGCTCT
GTATCGGGAA
120
GCCTTAGAGT
CTCCTGAGCA
TTGCTCACCT
CACCATACCG
CACTCAGGCA
AGCCATTCTC
180
TGCTGGGGGG
AATTGATGAC
TCTAGCTACC
TGGGTGGGTA
ATAATTTGGA
AGATCCAGCA
240
TCAAGGGACC
TAGTAGTCAA
TTATGTTAAT
ACTAACATGG
GTTTAAAAAT
TAGGCAACTA
300
TTGTGGTTTC
ATATATCTTG
CCTTACTTTT
GGAAGAGAGA
CTGTACTTGA
ATATTTGGTC
360
TCTTTCGGAG
TGTGGATTCG
CACTCCTCCA
GCCTATAGAC
CACCAAATGC
CCCTATCTTA
420
TCAACACTTC
CGGAAACTAC
TGTTGTTAGA
CGACGGGACC
GAGGCAGGTC
CCCTAGAAGA
480
AGAACTCCCT
CGCCTCGCAG
ACGCAGATCT
CAATCGCCGC
GTCGCAGAAG
ATCTCAATCT
540
CGGGAATCTC
AATGTTAG 558
__________________________________________________________________________
TABLE 2
__________________________________________________________________________
ATGGACATTG
ACCCTTATAA
AGAATTTGGA
GCTACTGTAG
AGTTACTCTC
GTTTTTGCCT
60
TCTGACTTCT
TTCCTTCCGT
CAGAGATCTC
CTAGACACCG
CCTCAGCTCT
GTATCGGGAA
120
GCCTTAGAGT
CTCCTGAGCA
TTGCTCACCT
CACCATACCG
CACTCAGGCA
AGCCATTCTC
180
TGCTGGGGGG
AATTGATGAC
TCTAGCTACC
TGGGTGGGTA
ATAATTTGGA
AGATCCAGCA
240
TCAAGGGATC
TAGTAGTCAA
TTATGTTAAT
ACTAACATGG
GTTTAAAAAT
TAGGCAACTA
300
TTGTGGTTTC
ATATATCTTG
CCTTACTTTT
GGAAGAGAGA
CTGTACTTGA
ATATTTGGCC
360
TCTTTCGGAG
TGTGGATTCG
CACTCCTCCA
GCCTATAGAC
CACCAAATGC
CCCTATCTTA
420
TCAACACTTC
CGGAAACTAC
TGTTGTTAGA
CGACGGGACC
GAGGCAGGTC
CCCTAGAAGA
480
AGAACTCCCT
CGCCTCGCAG
ACGCAGATCT
CAATCGCCGC
GTCGCAGAAG
ATCTCAATCT
540
CGGGAATCTC
AATGTTAG 558
__________________________________________________________________________
TABLE 3
__________________________________________________________________________
ATGGCTATTG
ACCCTTATAA
AGAATTTGGA
GCTACTGTGG
AGTTACTCTC
GTTTTTGCCT
60
TCTGACTTCT
TTCCTTCCGT
CAGAGATCTC
CTAGACACCG
CCTCAGCTCT
GTATCGGGAA
120
GCCTTAGAGT
CTCCTGAGCA
TTGCTCACCT
CACCATACCG
CACTCAGGCA
AGCCATTCTC
180
TCGTGGGGGG
AATTGATGAC
TCTAGCTACC
TGGGTGGGTA
ATAATTTGGA
AGATCCAGCA
240
TCAAGGGACC
TAGTAGTCAA
TTATGTTAAT
ACTAACATGG
GTTTAAAAAT
TAGGCAACTA
300
TTGTGGTTTC
ATATATCTTG
CCTTACTTTT
GGAAGAGAGA
CTGTACTTGA
ATATTTGGTC
360
TCTTTCGGAG
TGTGGATTCG
CACTCCTCCA
GCCTATAGAC
CACCAAATGC
CCCTATCTTA
420
TCAACACTTC
CGGAAACTAC
TGTTGTTAGA
CGACGGGACC
GAGGCAATCC
CCTAGAAGAA
480
GAACTCCCTC
GCCTCGCAGA
CGCAGATCTC
AATCGCCGCG
TCGCAGAAGA
TCTCAATCTC
540
GGGAATCTCA
ATGTTAGAAG
CTTCCGACAA
AACCGCCTAC
TCTCTTCTAA
AAGTCGGACT
600
ATGTCTAATT
TAGTCTTGCG
TCTTCGCCAG
ACTATTTTGT
CTTAA 645
__________________________________________________________________________
TABLE 4
__________________________________________________________________________
ATGGCTATTG
ACCCTTATAA
AGAATTTGGA
GCTACTGTGG
AGTTACTCTC
GTTTTTGCCT
60
TCTGACTTCT
TTCCTTCCGT
CAGAGATCTC
CTAGACACCG
CCTCAGCTCT
GTATCGGGAA
120
GCCTTAGAGT
CTCCTGAGCA
TTGCTCACCT
CACCATACCG
CACTCAGGCA
AGCCATTCTC
180
TGCTGGGGGG
AATTGATGAC
TCTAGCTACC
TGGGTGGGTA
ATAATTTGGA
AGATCCAGCA
240
TCAAGGGACC
TAGTAGTCAA
TTATGGTAAT
ACTAACATGG
GTTTAAAAAT
TAGGCAACTA
300
TTGTGGTTTC
ATATATCTTG
CCTTACTTTT
GGAAGAGAGA
CTGTACTTGA
ATATTTGGTC
360
TCTTTCGGAG
TGTGGATTCG
CACTCCTCCA
GCCTATAGAC
CACCAAATGC
CCCTATCTTA
420
TCAACACTTC
CGGAAACTAC
TGTTGTTAGA
CGACGGGACC
GAGGCAGGTC
CCCTAGAAGA
480
AGAACTCCCT
CGCCTCGCAG
ACGCAGATCT
CAATCGCCGC
GTCGCAGAAG
ATCTCAATCT
540
CGGGAATCTC
AATGTTAG 558
__________________________________________________________________________
TABLE 5
__________________________________________________________________________
ATGGCTATTG
ACCCTTATAA
AGAATTTGGA
GCTACTGTGG
AGTTACTCTC
GTTTTTGCCT
60
TCTGACTTCT
TTCCTTCCGT
CAGAGATCTC
CTAGACACCG
CCTCAGCTCT
GTATCGGGAA
120
GCCTTAGAGT
CTCCTGAGCA
TTGCTCACCT
CACCATACCG
CACTCAGGCA
AGCCATTCTC
180
TGCTGGGGGG
AATTGATGAC
TCTAGCTACC
TGGGTGGGTA
ATAATTTGGA
AGATCCAGCA
240
TCAAGGGACC
TAGTAGTCAA
TTATGTTAAT
ACTAACATGG
GTTTAAAAAT
TAGGCAACTA
300
TTGTGGTTTC
ATATATCTTG
CCTTACTTTT
GGAAGAGAGA
CTGTACTTGA
ATATTTGGCC
360
TCTTTCGGAG
TGTGGATTCG
CACTCCTCCA
GCCTATAGAC
CACCAAATGC
CCCTATCTTA
420
TCAACACTTC
CGGAAACTAC
TGTTGTTAGA
CGACGGGACC
GAGGCAGGTC
CCCTAGAAGA
480
AGAACTCCCT
CGCCTCGCAG
ACGCAGATCT
CAATCGCCGC
GTCGCAGAAG
ATCTCAATCT
540
CGGGAATCTC
AATGTTAG 558
__________________________________________________________________________
TABLE 6
__________________________________________________________________________
ATGGCTATTG
ACCCTTATAA
AGAATTTGGA
GCTACTGTGG
AGTTACTCTC
GTTTTTGCCT
60
TCTGACTTCT
TTCCTTCCGT
CAGAGATCTC
CTAGACACCG
CCTCAGCTCT
GTATCGGGAA
120
GCCTTAGAGT
CTCCTGAGCA
TTGCTCACCT
CACCATACCG
CACTCAGGCA
AGCCATTCTC
180
TGCTGGGGGG
AATTGATGAC
TCTAGCTACC
TGGGTGGGTA
ATAATTTGGA
AGATCCAGCA
240
TCAAGGGACC
TAGTAGTCAA
TTATGTTAAT
ACTAACATGG
GTTTAAAAAT
TAGGCAACTA
300
TTGTGGTTTC
ATATATCTTG
CCTTACTTTT
GGAAGAGAGA
CTGTACTTGA
ATATTTGGTC
360
TCTTTCGGAG
TGTGGATTCG
CACTCCTCCA
GCCTATAGAC
CACCAAATGC
CCCTATCTTA
420
TCAACACTTC
CGGAAACTAC
TGTTGTTAGA
CGACGGACCG
AGGCAGGTCC
CCTAGAAGAA
480
GAACTCCCTC
GCCTCGCAGA
CGCAGATCTC
AATCGCCGCG
TCGCAGAAGA
TCTCAATCTC
540
GGGAATCTCA
ATGTTAGAAG
CTTCCGACAA
AACCGCCTAC
TCTCTTCTAA
AAGTCGGACT
600
ATGTCTAATT
TAGTCTTGCG
TCTTCGCCAG
ACTATTTTGT
CTTAA 645
__________________________________________________________________________
TABLE 7
__________________________________________________________________________
##STR1##
##STR2##
##STR3##
##STR4##
##STR5##
##STR6##
##STR7##
##STR8##
##STR9##
##STR10##
##STR11##
##STR12##
__________________________________________________________________________
TABLE 8
__________________________________________________________________________
##STR13##
##STR14##
##STR15##
##STR16##
##STR17##
##STR18##
##STR19##
##STR20##
##STR21##
##STR22##
##STR23##
##STR24##
__________________________________________________________________________
TABLE 9
__________________________________________________________________________
##STR25##
##STR26##
##STR27##
##STR28##
##STR29##
##STR30##
##STR31##
##STR32##
##STR33##
##STR34##
##STR35##
##STR36##
__________________________________________________________________________
TABLE 10
__________________________________________________________________________
##STR37##
##STR38##
##STR39##
##STR40##
##STR41##
##STR42##
##STR43##
##STR44##
##STR45##
##STR46##
##STR47##
##STR48##
##STR49##
##STR50##
__________________________________________________________________________
TABLE 11
__________________________________________________________________________
##STR51##
##STR52##
##STR53##
##STR54##
##STR55##
##STR56##
##STR57##
##STR58##
##STR59##
##STR60##
##STR61##
##STR62##
##STR63##
##STR64##
__________________________________________________________________________
By only varying the number of bases (i.e., single nucleotides or base pairs
referring to pairs of complementary nucleotides) between the ribosomal
binding site and the start codon without the second codon change, an
increase in expression is also obtainable. Preferably the spacing (i.e.
spacer) between the ribosomal binding site and the start codon is between
8 and 20 nucleotides. Most preferably 10 to 15 nucleotides are between the
ribosomal binding site and the start codon. Sequence ID No: 13 and
Sequence ID No: 14 are good examples of a 10 base pair and 8 base pair
spacer, respectively. The particular nucleotide composition chosen to vary
the distance between the ribosomal binding site and the start codon can
vary. Preferably the nucleotides are chosen to have a high degree of
homology with the same region between the ribosomal binding sites and the
start codons of the host organisms.
Although the particular modification of the present invention at the second
codon utilizes GCT as the second codon, the particular modification of the
second codon is not limited to only GCT. For example, any codon utilized
by a large number of the host organisms can be engineered into the second
codon region. Preferred E. coli codons in the second position are AAA,
AGC, GCG, AAC, TCT, AAT, ACC, AGT, ACA, GCT, ACT, CAA, GCA, GAA, GGT and
ATC. It is unexpected that varying the second codon alone results in
increased expression. As evident from FIG. 1, changing the second codon
results in a two-fold increase in expression over the native sequence.
Since the modification of the gene at the second codon is especially
beneficial for enhancement and expression in the E. coli host, an E. coli
host is preferred.
Although any single alteration of the invention (e.g., spacer between the
ribosomal binding site and start codon, second codon change, and deletion
of basepair 455 or 468) in the DNA molecule results in increased
expression of about two (2) fold over the native sequence, if any two
alterations are made in combination, about a ten (10) fold increase in
expression is obtained. If all three alterations are made in combination,
about a twenty-five (25) fold increase in expression is obtained.
Importantly, the antigenicity of the HBcAg is not compromised by the
alterations. Even the deletion of base pair 455 or 468, which creates a
frame shift at the C-terminus of the HBcAg protein, does not alter the
antigenicity of the protein.
Sequence ID No: 1 was originally amplified from an HBsAg positive plasma
using appropriate primers and standard protocols. Now that the sequence is
known, the HBcAg sequence can be prepared in a variety of ways and
therefore is not limited to any particular preparation means.
For example, the nucleotide sequences of the invention can be prepared by
use of recombinant DNA technology or, alternatively, by automated
synthesis. The sequences of the invention can also be cloned in a suitable
vector and amplified in a suitable host.
Also, sequences of the invention can be synthesized using commercially
available methods and equipment. For example, the solid phase
phosphotriester method can be used to produce the sequences of the
invention. The sequences can be conveniently synthesized by the modified
phosphotriester method using fully protected DNA building blocks. Such
synthetic methods can be carried out in substantial accordance with the
procedure of Itakura, et al., 1977, Science 198:1056 and Crea, et al.,
1978, Proc. Nat. Acad. Sci. U.S.A., 75:575, and Narang, et al., 1980,
Methods in Enzymology, 68:90. In addition to manual procedures the
sequences can be synthesized using automated synthesizers.
Methods for solution and solid phase synthesis are widely known, and
various commercially available automatic synthesizers can be used in
accordance with known protocols. See, for example Stewart & Young, Solid
Phase Peptide Synthesis 2d Edition, Pierce Chemical Company, 1984; Tam, et
al., J. Am. Chem. Assoc. 105:6442 (1983) and Merrifield, et al.,
Biochemistry 21:5020 (1982), which are incorporated herein by reference.
The sequences of the invention can be produced by a number of procedures,
including synthetic DNA synthesis, cDNA cloning, genomic cloning,
polymerase chain reaction (PCR) technology, or a combination or these
approaches. See, e.g., Maniatis, MOLECULAR CLONING, A LABORATORY MANUAL,
1982. Mutagenesis procedures to produce "deletion mutants" that encode the
desired sequence can also be employed. Procedures suitable for producing
such mutants include polymerase chain reaction technology and variations
thereof, or site specific mutagenesis procedures similar to those of
Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985) or Eckstein, et al.,
Nucleic Acids Res. 13:8764 (1985).
For cloning, a selected DNA sequence of the invention will be inserted into
a suitable vector. A number of suitable vectors may be used, including
cosmids, plasmids, bacteriophage and viruses. The principal requirements
for such a vector are that it be capable of reproducing itself and stably
transforming a host cell. Most preferably, the vector will comprise an
"expression vector" which is capable of directing "expression" or cellular
production of a peptide encoded by the DNA sequence of the invention.
Typical expression vectors comprise a promoter region, a 5' untranslated
region, a coding sequence, a 3' untranslated region, an origin of
replication, a selective marker and a transcription termination site.
Suitable vectors for use in practicing the invention include pKK233-2
(Pharmacia), pKK223-2 (Pharmacia), pTTQ18 (Amersham, Arlington Heights,
Ill.), pBTacl (Boehringer Mannheim), pET expression systems (Novagen,
Madison, Wis.), and pPL-.lambda. (Pharmacia).
Promoters for use in expression vectors include, lactose (lac) control
elements, lambda (PL) control elements, arabinose control elements,
tryptophan (trp) control elements, and hybrids thereof. In addition, the
vector may contain any one of a number of various markers facilitating
selection of a transformed host cell. Such markers include genes
associated with temperature sensitivity, drug resistance (e.g., resistance
to ampicillin, chloramphenicol or tetracycline), or enzymes associated
with visual characeristics of the host organism.
The vector may be prepared for insertion of the DNA sequence in a number of
ways. Most simply, both the DNA sequence and the vector are digested with
an appropriate set of restriction enzymes to generate sites suitable for
ligation of the selected DNA sequence into the vector in an orientation
and position suitable to allow expression of the peptide encoded by the
sequence. Then, the DNA sequence is integrated into the vector by any of a
number of suitable procedures, including treatment with a selected ligase.
After the sequence has been integrated into the vector, the vector may be
used to transform a host cell. In general, the host cell may comprise any
cellular organism including a prokaryotic cell or eukaryotic cell that is
transformed with or competent of becoming transformed with the vector
comprising the sequences of the present invention. Suitable host cells
include, for example, bacterial cells such as E. coli or B. subtilis,
mammalian cells (Kaufman, High Level Production of Proteins in Mammalian
Cells, Genetic Engineering Principles and Methods, ed. J. K. Setlow Plenum
Press, 9:155, 1988), yeast (Barr, et al., Yeast Genetic Engineering, eds.
Butterworth, Boston, 1989), and insect cells (Maeda, Expression of Foreign
Genes in Insects using Baculovius Vectors, T. E. Mittler eds. Annual
Review of Entomology 34:351, 1989). A number of transformation techniques
suitable for use with the particular vector-host cell combination may be
employed. See, e.g., Maniatis, Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Laboratories (1982).
For production of HBcAg product, the transformed host cell is cultured in a
suitable medium under conditions designed to allow maximal expression with
the particular combination of host and vector employed. By transforming
hosts with sequences of the invention, greater amounts of HBcAg product is
obtained.
Sequences of the invention can be used in methods and kits designed to
detect the presence of antibodies in humans and therefore recognize
Hepatitis B virus (HBV) infected humans and blood samples which have been
infected by the hepatitis virus.
For example, the HBcAg produced by hosts transformed by recombinant DNA
molecules of this invention or synthetically, can be used in the
immunological diagnostic tests currently available for hepatitis B virus
detection, such as radioimmunoassay or ELISA (enzyme linked immunosorbent
assay). In one type of radioimmunoassay anti-core antigen antibody, raised
in a laboratory animal, is attached to a solid phase, such as the inside
of a test tube. HBcAg is then added to the tube so it can bind with the
antibody. To the tube coated with the antigen-antibody complex is added a
sample of the patient's serum, together with a known amount of HBV
anti-core antibody labelled with a radioactive isotope such as radioactive
iodine. Any HBV antibody in the patient's serum will compete with the
labelled antibody for the free binding sites on the antigen-antibody
complex. Once the serum has been allowed to interact, the excess liquid is
removed, the test tube washed, and the amount of radioactivity measured. A
positive result (i.e., that the patient's serum contains HBV antibody) is
indicated by a low radioactive count. In one type of ELISA test, a
microtitre plate is coated with HBcAg and a sample of a patient's serum is
added. After a period of incubation permitting interaction of any antibody
with the antigen, the plate is washed and a preparation of anti-human
antibodies, raised in a laboratory animal, and which are linked to an
enzyme label, is added, incubated to allow reaction to take place, and the
plate rewashed. Thereafter, enzyme substrate is added to the microtitre
plate and incubated for a period of time to allow the enzyme to work on
the substrate, and the adsorbance of the final preparation is measured. A
large change in adsorbance indicates a positive result.
The following examples illustrate the specific embodiments of the invention
described in this document. As would be apparent to skilled artisans,
various changes and modifications are possible and are contemplated within
the scope of the invention described.
EXAMPLES
Materials and Preparation
Amplification of Core Gene
Polymerase chain reactions (PCR) are set up as follows:
______________________________________
H.sub.2 O 21.5 .mu.l
PCR 10X Buffer* 10.0 .mu.l
1.25 mM dNTPs 16.0 .mu.l
20 .mu.M Forward Primer
1.0 .mu.l
20 .mu.M Reverse Primer
1.0 .mu.l
HBV DNA 50.0 .mu.l
P/E Amplitaq 0.5 .mu.l
100.0 .mu.l
*PCR 10X Buffer:
30 cycles:
95 degrees C. - 2.0 minutes
500 mM KCl 50 degrees C. - 1.5 minutes
100 mM Tris-HCl (pH 8.3)
72 degrees C. - 1.5 minutes
15 mM MgCl.sub.2
0.1% gelatin
______________________________________
Oligonucleotide Primer Design and Synthesis
Oligonucleotide primers are synthesized on an Applied Biosystems, Inc.
(ABI) DNA Synthesizer Model 381-A (Foster City, Calif.), or equivalent,
according to manufacturer's specifications. "Trityl-On" oligonucleotides
are purified using ABI "OPC" purification columns or equivalent according
to manufacturer's protocol. One ml of eluent is dried in a Savant
Speed-Vac, or equivalent, and resuspended in 100 .mu.l of 10 mM Tris-HCl
(pH 7.2), 1 mM EDTA. A 20 .mu.M Stock solution of each primer is made for
use in the PCR.
The following is the list of oligo primers for amplifying the core gene for
cloning and expression.
__________________________________________________________________________
Forward primers for expression in pKK233
"native"
(Seq ID No: 15) GGCC ATG GAC ATT GAC CCT TAT AAA
2nd codon
##STR65##
GGA
Forward primers for expression in pKK223
EcoRI
10 bp spacer
(Seq ID No: 17) CCGAATTC ATG GAC ATT GAC CCT TAT AAA
2nd + 10 bp
##STR66##
11 bp (Seq ID No: 19) CGGAATTCG ATG GAC ATT GAC CCT TAT AAA
12 bp (Seq ID No: 20) CGGAATTCGG ATG GAC ATT GAC CCT TAT AAA
13 bp (Seq ID No: 21) CGGAATTCGGA ATG GAC ATT GAC CCT TAT AAA
14 bp (Seq ID No: 22) CGGAATTCGGAT ATG GAC ATT GAC CCT TAT AAA
15 bp (Seq ID No: 23) CGGGAATTCGGATC ATG GAC ATT GAC CCT TAT AAA
Reverse primer for both pKK233 and pKK223
HindIII
(Seq ID No: 24) GGAAGCTT CTA ACA TTG AGA TTC CCG AGA TTG
AGA TCT TCT GCG
__________________________________________________________________________
EXAMPLE 1
Isolation of Hepatitis B DNA
Using a protocol in substantial accordance with I. Baginski, et. al.
"Detection of Hepatitis B Virus", In PCR Protocols, edited by M. A. Innis,
D. H. Gelfand, J. J. Sninsky and T. J. White, 348-355, Academic Press,
Inc. (1990). Hepatitis B virus (HBV) DNA is isolated from an HBsAg
positive serum (sample #2791-19A obtained from Interstate Blood Bank,
Inc., Memphis, Tenn.) as follows:
To about 60 .mu.l of HBsAg positive serum is added about 75 .mu.l of 250
.mu.g/ml proteinase K (Boehringer Mannheim, Indianapolis, Ind.) in: 0.25%
SDS, 5 mM EDTA, 10 mM Tris-HCl (pH 8.0). This solution is incubated for
about 2 hours at about 56 degrees C. The proteinase K is then heat
inactivated at about 95 degrees C. for about 10 minutes. The total volume
is brought to 1215 .mu.l in 1X Taq Polymerase buffer: 50 mM KCl, 10 mM
Tris-HCl (pH 8.4), 2.5 mM MgCl.sub.2, 0.01% gelatin (w/v), 0.5% Tween 20,
0.5% NP 40. Fifty microliters of this prepared DNA is used in subsequent
polymerase chain reactions (PCRs) for generating HBcAg clones.
EXAMPLE 2
Cloning of Amplified HBcAg Gene Fragment
A PCR is set up using primers Sequence ID No: 15 and Sequence ID No: 24 to
generate the first core gene fragment to be cloned. The resulting
.about.570 bp fragment and the expression vector pKK233-2 (Pharmacia) are
doubly digested with NcoI/HindIII (BRL). A ligation is set up with T4
ligase (BRL). Using a method in substantial accordance witin the teaching
of D. H. Hanahan, "Techniques for Transformation of E. coli", In DNA
cloning volume I, a practical approach, edited by D. M. Glover, 109, IRL
Press Limited (1985), frozen competent E. coli strain XL1Blue (Stratagene)
is transformed with the ligation, spreading 100 .mu.l of the
transformation mix on LB (100 .mu.g/ml ampicillin) plates. Transformants
are screened for inserts and those containing the desired fragment are
used in expression experiments.
EXAMPLE 3
Expression of Core Protein
Cultures (12.5 ml) LB (100 .mu.g/ml ampicillin) are grown to mid log then
induced with isopropylthio-.beta.-D-galactoside (IPTG) to a final
concentration of 1 mM. Cultures are allowed to grow overnight,
approximately 16 hours. 1.5 ml of the culture is removed, spun down at
full speed in a Brinkman microfuge or equivalent. The pellet is
resuspended in 100 .mu.l 1X sample prep buffer (U. K. Laemmli, Nature
227:680 (1970), boiled about 5 minutes and 10 .mu.l run on a 12% SDS PAGE
gel at 40 mA for about 11/4 hour. A Western blot is prepared by
transferring proteins from the gel to Immobilon-P membrane (Millipore,
Bedford, Mass.), or equivalent at 100 mA in a Hoefer Semidry Transfer
apparatus or equivalent for 1 hour using Towbin buffer (25 mM Tris (pH
8.3), 192 mM Glycine, 15% methanol). After transfer, membrane is blocked
with 5% BSA in 1X Tris buffered saline (TBS) (10 mM Tris-HCl, (pH 8.0),
0.9% NaCl) for 30 minutes. An anti-HBcAg polyclonal antibody (this
antibody solution in phosphate buffered saline (PBS)+a carrier protein, is
a component of a kit, but is sold separately, this antibody concentration
is unknown) (BioGenex Laboratories, Dublin, Calif.--catalog #PA082-5P) is
incubated with the membrane for 1 hour. Membrane is washed 5X with TBS.
Membrane was then incubated with a goat anti-rabbit horseradish peroxidase
conjugated polyclonal antibody (Cappel Organonteknika, West Chester, Pa.)
at 1:1000 in the 5% bovine serum albumin (BSA)/TBS solution for one hour.
Membrane is washed 5X with TBS then developed with 4-chloro-1-naphthol and
hydrogen peroxide. Clones expressing HBcAg show the appropriate size
reactive band at .about.21 KDa. However, all show very low levels of
expression.
EXAMPLE 4
Increasing Expression Levels
Oligonucleotide primers are synthesized (see "Materials and Preparations"
above) to allow about 10 to 15 bp between the ribosome binding site and
the ATG start codon. PCRs are set up with the following primer sets using
the method previously described: Sequence ID No: 17--Sequence ID No: 24,
Sequence ID No: 19--Sequence ID No: 24, Sequence ID No: 20--Sequence ID
No: 24, Sequence ID No: 21--Sequence ID No: 24, Sequence ID No:
22--Sequence ID No: 24, and Sequence ID No: 23--Sequence ID No: 24. All
PCRs generate the appropriate .about.570 bp fragment. These fragments are
doubly digested with EcoRI and Hind III and directionally cloned into
pKK223-3 (Pharmacia, Piscataway, N.J.) as described above. Transformations
are set up as previously described.
LB plates are spread with 50 .mu.l of 1M IPTG and a membrane sandwich
placed on the surface of the agar. The sandwich is prepared by placing a
nitrocellulose membrane (BA 85; Schleicher & Schuell, Inc., Keene, N.H.)
or equivalent, on top of the agar medium and then a cellulose acetate
membrane (OE67; Schleicher & Schuell), or equivalent, on top of that.
Transformations are then plated as before and plates incubated at about 37
degrees C. overnight. The nitrocellulose filters are removed and blocked
for about 1 hour in 5% BSA/TBS. An anti-HBcAg monoclonal antibody is added
at 20 .mu.g/ml and incubated for about 1 hour. Membranes are washed with
TBS a secondary goat anti-mouse horseradish conjugated antibody (Cappel)
is added at 1:2000 in 5% BSA/TBS for about 1 hour. Membranes are washed
with TBS and developed with 4-chloro-1-naphthol and hydrogen peroxide.
Reactive colonies are patched out for a second direct colony immunoblot
(DCI) and again chosen for reactivity. The 10 bp spacer clones are chosen
for further analysis and quantitation in a sandwich ELISA.
Two oligo primers Sequence ID No: 16 and Sequence ID No: 18 (see "materials
and Preparation",) are designed to change the HBcAg second codon from GAC
to GCT. Primer Sequence ID No: 16 changes the second codon with the
fragment to be cloned into pKK233-2 while primer Sequence ID No: 18 allows
for the 10 bp spacer as well as the second codon change. PCRs are set up
and fragments cloned as previously mentioned. DCI are set up for screening
transformants. Reactive colonies are chosen for further analysis and
quantitation in a sandwich ELISA.
EXAMPLE 5
Quantitation for Increased Expression of HBcAg
Strains tested by ELISA
______________________________________
Strain Vector Fragment
______________________________________
Sequence ID No: 1
pKK233-2 HBcAg DNA sequence
Sequence ID No: 3
pKK223-3 10 bp spacer
Sequence ID No: 5
Pkk223-3 10 bp spacer +
2nd codon change
Control pKK223-3 No insert
______________________________________
3 ml overnight cultures (LB--100 .mu.g/ml Ampicillin) of each of the above
stains are grown at about 37 degrees C. 250 .mu.l of the overnight culture
(1:50) is used to inoculate 12.5 ml cultures (same medium). Cultures are
grown to 0.6-0.8 A.sub.600 then induced by adding 1M IPTG to .about.1 mM.
Cultures are allowed to grow overnight (about 19 hours). One O.D. unit
(A.sub.600) of cells from each culture is removed and spun down and
resuspended in 975 ul of PBS to which 10 .mu.l of 10 .mu.g/ml lysozyme
(Sigma, St. Louis, Mo.) in PBS was added. Each is frozen in liquid
nitrogen and thawed at about 37 degrees C. 3 times. 1M MgCl.sub.2 is added
to 5 mM (5 .mu.l) and 10 .mu.l of 1 mg/ml DNase (Sigma) is added and then
the solution is incubated at room temperature for about 10 minutes.
Lysates are spun at .about.7000 xg in microfuge for about 10 minutes. The
supernatant fraction is removed and used in the ELISA.
One hundred microliters of "capture" antibody is coated onto plates at 20
.mu.g/ml in phosphate buffered saline (PBS) (10 mM Phosphate (pH 7.2), 130
mM NaCl) at about 4 degrees C. overnight. Capture antibody solution is
removed and the wells blocked with 2.5% BSA in PBS for about 1 hour. Fifty
microliters of serial two fold dilutions of E. coli lysates (in 2.5%
BSA/PBS) from 1:8 down to 1:16,000 are placed in wells and incubated for
about 1 hour. Wells are washed 5 times with PBS+0.05% Tween 20
(PBS-Tween). Fifty microliters of biotinylated detector antibody is added
at 5 .mu.g/ml in 2.5% BSA/PBS to each well and incubated for about 1 hour.
Plates are washed 5 times with PBS-Tween. Fifty microliters of Avidin-HRP
(1 mg/ml) (Sigma) diluted 1:2000 in 2.5% BSA/PBS are added to each well
and incubated for about 1 hour. Plates are washed 5 times with PBS Tween,
and developed with 50 .mu.l of o-phenylenediamine dihydrochloride (OPD)
(20 mg OPD, 100 mM citrate (pH 5.5), 7 .mu.l 30% hydrogen peroxide). After
two minutes the reaction is stopped by adding about 50 .mu.l of 4.5M
sulphuric acid. Plates are read at A.sub.490. Relative quantities of HBcAg
in each lysate is calculated against a standard curve generated with
purified HBcAg from 20 .mu.g to 0.02 ng per well (2 fold serial dilution).
Although the invention has been described with respect to specific
modifications, the details thereof are not to be construed as limitations,
for it will be apparent that various equivalents, changes and
modifications may be resorted to without departing from the spirit and
scope thereof, and it is understood that such equivalent embodiments are
to be included therein.
__________________________________________________________________________
SEQUENCELISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 24
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 558 base pairs
(B) TYPE: nucleic acid
(C ) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..558
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC48
MetAspIleAspProTyrLysGluPheGl yAlaThrValGluLeuLeu
151015
TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC96
SerPheLeuProSerAspPhePheProS erValArgAspLeuLeuAsp
202530
ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC144
ThrAlaSerAlaLeuTyrArgGluAlaLeu GluSerProGluHisCys
354045
TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAA192
SerProHisHisThrAlaLeuArgGlnAlaIleLeu CysTrpGlyGlu
505560
TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspPr oAla
65707580
TCAAGGGACCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAA288
SerArgAspLeuValValAsnTyrValAsnThrAsnMetG lyLeuLys
859095
ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThr PheGlyArg
100105110
GAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACT384
GluThrValLeuGluTyrLeuValSerPheGlyValTrpIle ArgThr
115120125
CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432
ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPr o
130135140
GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480
GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg
145 150155160
AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528
ArgThrProSerProArgArgArgArgSerGlnSerProArgArgArg
165170175
AGATCTCAATCTCGGGAATCTCAATGTTAG558
ArgSerGlnSerArgGluSerGlnCys
180 185
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu
1 51015
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ThrAlaSerAlaLeuTyrArgGlu AlaLeuGluSerProGluHisCys
354045
SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu
505560
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys
859095
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105110
GluThrValLeuG luTyrLeuValSerPheGlyValTrpIleArgThr
115120125
ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro
130135 140
GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg
145150155160
ArgThrProSerProArgArgArgArgSerGlnSerPro ArgArgArg
165170175
ArgSerGlnSerArgGluSerGlnCys
180185
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 558 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..558
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATGGACATTGACCCTTATAAAGAATTTGGAGCTACTGTAGAGTTACTC48
MetAspIleAs pProTyrLysGluPheGlyAlaThrValGluLeuLeu
151015
TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC96
SerPheLeuP roSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC144
ThrAlaSerAla LeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAA192
SerProHisHisThrAla LeuArgGlnAlaIleLeuCysTrpGlyGlu
505560
TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240
LeuMetThrLeuAlaThrTrpValGl yAsnAsnLeuGluAspProAla
65707580
TCAAGGGATCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAA288
SerArgAspLeuValValAsnT yrValAsnThrAsnMetGlyLeuLys
859095
ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336
IleArgGlnLeuLeuTrpPhe HisIleSerCysLeuThrPheGlyArg
100105110
GAGACTGTACTTGAATATTTGGCCTCTTTCGGAGTGTGGATTCGCACT384
GluThrValLeuGluTyrLeuAla SerPheGlyValTrpIleArgThr
115120125
CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432
ProProAlaTyrArgProProAsnAlaPr oIleLeuSerThrLeuPro
130135140
GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480
GluThrThrValValArgArgArgAspArgGlyArgS erProArgArg
145150155160
AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528
ArgThrProSerProArgArgArgArgSerGln SerProArgArgArg
165170175
AGATCTCAATCTCGGGAATCTCAATGTTAG558
ArgSerGlnSerArgGluSerGlnCys
180185
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
MetAspIleAspProTyrLysGluPheGlyAlaThrValGluLe uLeu
151015
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ThrAla SerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu
50 5560
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
SerArgAspLeuValValAsnTyrValAsnThr AsnMetGlyLeuLys
859095
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105 110
GluThrValLeuGluTyrLeuAlaSerPheGlyValTrpIleArgThr
115120125
ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro
130 135140
GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg
145150155160
ArgThrProSerProArgArg ArgArgSerGlnSerProArgArgArg
165170175
ArgSerGlnSerArgGluSerGlnCys
180185
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 645 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..645
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATGGCTATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC 48
MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu
151015
TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC 96
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC 144
ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTCGTGGGGGGAA192
SerProHisHisThrAlaLeuArgGlnAlaIleLeuSerTrpGlyGlu
505560
TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240
LeuMetTh rLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
TCAAGGGACCTAGTAGTCAATTATGTTAATACTAACATGGGTTTAAAA288
SerA rgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys
859095
ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336
Ile ArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105110
GAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACT384
GluThr ValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr
115120125
CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432
ProProAlaTy rArgProProAsnAlaProIleLeuSerThrLeuPro
130135140
GAAACTACTGTTGTTAGACGACGGGACCGAGGCAATCCCCTAGAAGAA480
GluThrThrValValArgA rgArgAspArgGlyAsnProLeuGluGlu
145150155160
GAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGAA528
GluLeuProArgLeu AlaAspAlaAspLeuAsnArgArgValAlaGlu
165170175
GATCTCAATCTCGGGAATCTCAATGTTAGAAGCTTCCGACAAAACCGC576
AspLeuAsnLeuGly AsnLeuAsnValArgSerPheArgGlnAsnArg
180185190
CTACTCTCTTCTAAAAGTCGGACTATGTCTAATTTAGTCTTGCGTCTT624
LeuLeuSerSerLysSe rArgThrMetSerAsnLeuValLeuArgLeu
195200205
CGCCAGACTATTTTGTCTTAA645
ArgGlnThrIleLeuSer
210215
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu
151015
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ThrAlaSerAl aLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
SerProHisHisThrAlaLeuArgGlnAlaIleLeuSerTrpGlyGlu
5055 60
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
SerArgAspLeuValValAsnTyrValAsnThrAsnM etGlyLeuLys
859095
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105110
GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr
115120125
ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro
130 135140
GluThrThrValValArgArgArgAspArgGlyAsnProLeuGluGlu
145150155160
GluLeuProArgLeuAlaAspAlaAs pLeuAsnArgArgValAlaGlu
165170175
AspLeuAsnLeuGlyAsnLeuAsnValArgSerPheArgGlnAsnArg
180185 190
LeuLeuSerSerLysSerArgThrMetSerAsnLeuValLeuArgLeu
195200205
ArgGlnThrIleLeuSer
210
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 558 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..558
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
ATGGCTATTGACCCTTATAAAGAATTTGGAGCTACTGTGGAGTTACTC 48
MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu
151015
TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC 96
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTCTCCTGAGCATTGC1 44
ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCTGGGGGGAA192
Ser ProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu
505560
TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA240
LeuMetThrLe uAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
TCAAGGGACCTAGTAGTCAATTATGGTAATACTAACATGGGTTTAAAA288
SerArgA spLeuValValAsnTyrGlyAsnThrAsnMetGlyLeuLys
859095
ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGGAAGA336
IleArg GlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105110
GAGACTGTACTTGAATATTTGGTCTCTTTCGGAGTGTGGATTCGCACT384
GluThrVal LeuGluTyrLeuValSerPheGlyValTrpIleArgThr
115120125
CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG432
ProProAlaTyrAr gProProAsnAlaProIleLeuSerThrLeuPro
130135140
GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480
GluThrThrValValArgArgA rgAspArgGlyArgSerProArgArg
145150155160
AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528
ArgThrProSerProArg ArgArgArgSerGlnSerProArgArgArg
165170175
AGATCTCAATCTCGGGAATCTCAATGTTAG558
ArgSerGlnSerArgGlu SerGlnCys
180185
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
MetAlaIleAspProTyrLysGluPheGl yAlaThrValGluLeuLeu
151015
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
2025 30
ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu
505560
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
SerArgAspLeuValVal AsnTyrGlyAsnThrAsnMetGlyLeuLys
859095
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100 105110
GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr
115120125
ProProAlaTyrArgProProAsnAlaProIleLeuSerTh rLeuPro
130135140
GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg
145150155160
ArgThr ProSerProArgArgArgArgSerGlnSerProArgArgArg
165170175
ArgSerGlnSerArgGluSerGlnCys
180185
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 558 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..558
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATGGCTATTGACCCTTATAAAGAATTTGGAGCT ACTGTGGAGTTACTC48
MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu
151015
TCGTTTTTGCCTTCTGACTTCTTTCCTTCCGTC AGAGATCTCCTAGAC96
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ACCGCCTCAGCTCTGTATCGGGAAGCCTTAGAGTC TCCTGAGCATTGC144
ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
TCACCTCACCATACCGCACTCAGGCAAGCCATTCTCTGCT GGGGGGAA192
SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu
505560
TTGATGACTCTAGCTACCTGGGTGGGTAATAATTTGGAAGATCCAGCA 240
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
TCAAGGGACCTAGTAGTCAATTATGTTAATACTAACATGGGTTTA AAA288
SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys
859095
ATTAGGCAACTATTGTGGTTTCATATATCTTGCCTTACTTTTGG AAGA336
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105110
GAGACTGTACTTGAATATTTGGCCTCTTTCGGAGTGTGGATTCGCA CT384
GluThrValLeuGluTyrLeuAlaSerPheGlyValTrpIleArgThr
115120125
CCTCCAGCCTATAGACCACCAAATGCCCCTATCTTATCAACACTTCCG 432
ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro
130135140
GAAACTACTGTTGTTAGACGACGGGACCGAGGCAGGTCCCCTAGAAGA480
GluT hrThrValValArgArgArgAspArgGlyArgSerProArgArg
145150155160
AGAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGCGTCGCAGA528
ArgThrProSerProArgArgArgArgSerGlnSerProArgArgArg
165170175
AGATCTCAATCTCGGGAATCTCAATGTTAG558
ArgSerGlnSerArgGluSerGlnCys
180185
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
MetAlaIleAs pProTyrLysGluPheGlyAlaThrValGluLeuLeu
151015
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
20 2530
ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
SerProHisHisThrAlaLeuArgGlnAlaIleL euCysTrpGlyGlu
505560
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys
859095
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105110
GluThrValLeuGluTyrLeuAlaSerPheGlyValTrpIleArgThr
115120125
ProProAlaTyrArgProProAs nAlaProIleLeuSerThrLeuPro
130135140
GluThrThrValValArgArgArgAspArgGlyArgSerProArgArg
145150155 160
ArgThrProSerProArgArgArgArgSerGlnSerProArgArgArg
165170175
ArgSerGlnSerArgGluSerGlnCys
180 185
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 645 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..645
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATGGCTATTGACCCTT ATAAAGAATTTGGAGCTACTGTGGAGTTACTC48
MetAlaIleAspProTyrLysGluPheGlyAlaThrValGluLeuLeu
151015
TCGTTTTTGCCTTCT GACTTCTTTCCTTCCGTCAGAGATCTCCTAGAC96
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
202530
ACCGCCTCAGCTCTGTAT CGGGAAGCCTTAGAGTCTCCTGAGCATTGC144
ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
TCACCTCACCATACCGCACTCAG GCAAGCCATTCTCTGCTGGGGGGAA192
SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysTrpGlyGlu
505560
TTGATGACTCTAGCTACCTGGGTGGGTAATA ATTTGGAAGATCCAGCA240
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
TCAAGGGACCTAGTAGTCAATTATGTT AATACTAACATGGGTTTAAAA288
SerArgAspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys
859095
ATTAGGCAACTATTGTGGTTTCATATA TCTTGCCTTACTTTTGGAAGA336
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100105110
GAGACTGTACTTGAATATTTGGTCTCTTT CGGAGTGTGGATTCGCACT384
GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr
115120125
CCTCCAGCCTATAGACCACCAAATGCCCCTATCT TATCAACACTTCCG432
ProProAlaTyrArgProProAsnAlaProIleLeuSerThrLeuPro
130135140
GAAACTACTGTTGTTAGACGACGGACCGAGGCAGGTCCCCTA GAAGAA480
GluThrThrValValArgArgArgThrGluAlaGlyProLeuGluGlu
145150155160
GAACTCCCTCGCCTCGCAGACGCAGATCTCAATCGCCGC GTCGCAGAA528
GluLeuProArgLeuAlaAspAlaAspLeuAsnArgArgValAlaGlu
165170175
GATCTCAATCTCGGGAATCTCAATGTTAGAAGCTTCCG ACAAAACCGC576
AspLeuAsnLeuGlyAsnLeuAsnValArgSerPheArgGlnAsnArg
180185190
CTACTCTCTTCTAAAAGTCGGACTATGTCTAATTTAGTCT TGCGTCTT624
LeuLeuSerSerLysSerArgThrMetSerAsnLeuValLeuArgLeu
195200205
CGCCAGACTATTTTGTCTTAA 645
ArgGlnThrIleLeuSer
210215
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
MetAlaIleAspProTy rLysGluPheGlyAlaThrValGluLeuLeu
151015
SerPheLeuProSerAspPhePheProSerValArgAspLeuLeuAsp
20 2530
ThrAlaSerAlaLeuTyrArgGluAlaLeuGluSerProGluHisCys
354045
SerProHisHisThrAlaLeuArgGlnAlaIleLeuCysT rpGlyGlu
505560
LeuMetThrLeuAlaThrTrpValGlyAsnAsnLeuGluAspProAla
65707580
SerArg AspLeuValValAsnTyrValAsnThrAsnMetGlyLeuLys
859095
IleArgGlnLeuLeuTrpPheHisIleSerCysLeuThrPheGlyArg
100 105110
GluThrValLeuGluTyrLeuValSerPheGlyValTrpIleArgThr
115120125
ProProAlaTyrArgProProAsnAlaPr oIleLeuSerThrLeuPro
130135140
GluThrThrValValArgArgArgThrGluAlaGlyProLeuGluGlu
145150155 160
GluLeuProArgLeuAlaAspAlaAspLeuAsnArgArgValAlaGlu
165170175
AspLeuAsnLeuGlyAsnLeuAsnValArgSerPheArgGlnAsnArg
180185190
LeuLeuSerSerLysSerArgThrMetSerAsnLeuValLeuArgLeu
195200205
ArgGlnThrIleLeuSer
210
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
AACAGAATTC10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
AACAGACC8
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GGCCATGGACATTGACCCTTATAAA25
(2) INFORMATION FOR SEQ ID NO:16:
( i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GGCCATGGTCATTGACCCTTATAAAGAATTTGGA34
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CCGAATTCATGGACATTGACCCTTATAAA29
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CCGAATTCATGGTCATTGACCCTTATAAA29
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CGGAATTCGATGGACATTGACCCTTATAAA30
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CGGAATTCGGATGGACATTGACCCTTATAAA31
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CGGAATTCGGAATGGACATTGACCCTTATAAA32
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CGGAATTCGGATATGGACATTGACCCTTATAAA33
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D ) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CGGGAATTCGGATCATGGACATTGACCCTTATAAA35
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GGAAGCTTCTAACATTGAGATTCCCGAGATTGAGATCTTCTGCG44
Top